Polyketides are widely used in human and veterinary medicine as antibiotic, immunosuppressant, antitumor, antifungal, and antiparasitic agents, the discovery of new members of this class and understanding how these compounds are biosynthesized is of paramount importance. Besides the practical value of such studies, the study of polyketide synthase enzymology is also of fundamental significance. Indeed, modular polyketide synthases are among the largest and most complex biological catalysts that are known. Using a small suite of biochemical reactions that are repeated in a tightly programmed manner, these synthases carry out the non-templated, assembly line synthesis of some of the most structurally and stereochemically complex natural products. The unusual size (2-5 MDa) of these multifunctional synthases and the fact that all of their intermediates remain covalently tethered to the acyl carrier protein components throughout the entire reaction cycle makes the study of these extraordinary enzymes especially challenging and scientifically rewarding. The proposed studies will lead to fundamental new biological insights into the central issue of modern molecular biochemistry, the relationship between protein sequence, structure, and function. During the next project period, we will continue to investigate the mechanistic enzymology and molecular genetics of complex polyketide natural product biosynthesis. We will use a combination of chemical, enzymological, protein engineering, and protein structural approaches to elucidate the individual mechanisms and the molecular basis for the programming of the multistep, enzyme-catalyzed transformations that lead to the formation of complex polyketides, including the macrolide antibiotics erythromycin, methymycin, picromycin, tylosin, and rifamycin, the polyene antifungal agent nystatin, the protein phosphatase 2A inhibitor fostriecin, and the polyether nanchangmycin. The core experimental approach will be based on the expression and characterization of individual PKS domains and modules and the use of these recombinant proteins, alone or in combination, to establish the intimate biochemical details of reaction mechanism and protein-protein recognition in polyketide biosynthesis. Special emphasis will be placed on 1) understanding the mechanism, substrate specificity, and stereochemistry of the reduction reactions catalyzed by ketoreductase (KR) domains; 2) confirming the recently demonstrated role of specific KR domains in the epimerization of methyl groups during the formation of reduced polyketides and identifying the source of epimerization of unreduced polyketide intermediates; 3) understanding the mechanism, substrate specificity, and stereochemistry of the dehydration reactions catalyzed by dehydratase (DH) domains, both alone and in intact modules; and 4) elucidating the complex relationships between PKS structure and function. PUBLIC HEALTH RELEVANCE: Polyketides are widely used in human and veterinary medicine as antibiotic, immunosuppressant, antitumor, antifungal, and antiparasitic agents. The discovery of new members of this class and understanding how these compounds are biosynthesized is of paramount importance. Besides the practical value of such studies, the study of polyketide synthase enzymology is also of fundamental significance. Indeed, modular polyketide synthases are among the largest and most complex biological catalysts that are known. Using a small suite of biochemical reactions that are repeated in a tightly programmed manner, these synthases carry out the non-templated, assembly line synthesis of some of the most structurally and stereochemically complex natural products. The unusual size (2-5 MDa) of these multifunctional synthases and the fact that all of their intermediates remain covalently tethered to the acyl carrier protein components throughout the entire reaction cycle makes the study of these extraordinary enzymes especially challenging and scientifically rewarding. Finally, the proposed studies will lead to fundamental new biological insights into the central issue of modern molecular biochemistry, the relationship between protein sequence, structure, and function.